This invention relates to mutants of atrial natriuretic factor, particularly mutants of atrial natriuretic factor that are protective for stroke. The invention further relates to methods of treating or preventing stroke by administration of mutants of atrial natriuretic factor. Additionally, the invention relates to methods of diagnosis, prognosis and screening for alleles of the atrial natriuretic factor gene that are protective for stroke.
Ischemic stroke is a common, complex disorder caused by a combination of genetic and environmental factors that is associated with long term disability and death. Apart from the treatment of hypertension, a risk factor for stroke (Kannel et al., JAMA 214:301-310 (1970); Veterans Administration Cooperative Study Group on Antihypertensive Agents, Effects of treatment of morbidity in hypertension, JAMA 213:1143-1152 (1970)), few preventative therapeutics are available, and no genetic test for stroke predisposition exists. A significant genetic component to human stroke predisposition has been demonstrated by both rare monogenic inheritance (Palsdottir et al. Lancet 2:603-604 (1988); Boers et al., N. Eng. J. Med. 313:709-715 (1985); Levy et al., Science 248:1124-1126 (1990)) and by increased concordance in monozygotic compared with dizygotic twins (De Faire et al., Prev. Med. 4:509-517 (1975); Brass et al. Stroke 23:221-223 (1992)).
The spontaneously hypertensive stroke-prone rat (SHRSP) strain is an animal model of ischemic stroke. SHRSP rats suffer cerebrovascular events with 99% penetrance when administered a diet high in sodium to induce hypertension. These SHRSP rats are closely related to the SHR (spontaneously hypertensive rat) strain which develops hypertension, but not ischemic stroke, when administered a high sodium diet. Genetic analysis of the progeny of SHR-SHRSP rat intercrosses revealed that three Quantitative Trait Loci (QTLS) contribute to stroke latency (Rubattu et al., Nat. Gen. 13:429-434 (1996)). While a locus on chromosome 1 significantly correlated with the occurrence of stroke, two loci, one on chromosome 4 and another on chromosome 5, were actually associated with increased stroke latency. The chromosome 5 locus mapped near the gene coding for ANF (atrial natriuretic factor) and BNF (brain natriuretic factor). None of the three loci were associated with hypertension.
A second study by Jeffs et al. (Nat. Gen. 16:364-367 (1997)) analyzed F2 progeny of intercrosses between the SHRSP animals and a normotensive, non-stroke rat strain Wistar-Kyoto (WKY). A chromosome 5 locus, which also co-localized with ANF and BNF, was implicated in increased infarct susceptibility and severity in the SHRSP rats.
ANF is a small peptide hormone known to have vasoactive and diuretic activities and functions both to relax smooth muscle and to reduce sodium reabsorption and intravascular volume (Needleman et al., Ann. Rev. Pharmacol. Toxicol. 29:23-54 (1989)). ANF is derived from the prohormone atrial natriuretic peptide (ANP) which appears to be expressed in response to increased blood volume or sodium levels. The proANP is initially expressed as a single polypeptide chain and is proteolytically processed within the cell into four peptides, all of which have been implicated in the diuretic process of vasodilation: Long Acting Natriuretic Peptide, Vessel Dialator, Kaliuretic Peptide and Atrial Natriuretic Factor (Vesely et al., Metabolism: Clinical and Experimental 45: 315-319 (1996)). ANF is derived from amino acids 99-126 of ProANP. The physiological activity of ANF appears to be mediated through binding to the guanylate cyclase-A receptor by increasing secretion of cGMP into plasma and urine (Vesely et al., Amer. J. Med. Sci. 310:143-149 (1995); Miao et al., Hypertension 25:694-69 (1995)).
Garcia et al. (Hypertension 13:567-574 (1989)) previously found that SHR had higher plasma ANF levels, and lower glomerular ANF receptor density, than normotensive controls. Additionally, Estrada et al. (Am. J. Hypertens. 7:1085-1089 (1994)) noted that elevated ANF levels have been detected in ischemic stroke patients. However, there has been no suggestion in the art that mutant ANF proteins or modulators (e.g., antagonists, agonists or inhibitors) of ANF would be useful as therapeutics or prophylactics for ischemic stroke.
The present inventors have discovered that the ANF gene in the SHRSP strain has the glycine substituted with a serine residue at position 1 of rat ANF as depicted in FIG. 2 (SEQ ID NO:1) (amino acid 99 of the proANP amino acid sequence as depicted in FIG. 3 (SEQ ID NO:2)). Furthermore, the present inventors have realized that this mutant ANF locus coincides with the chromosomal locus associated with increased stroke latency in the SHRSP animals. Accordingly, the ANF mutants and mutant ANF alleles described herein are useful in the treatment and prevention of ischemic stroke, and screening for a genetic predisposition for protection against ischemic stroke.
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.
The present invention relates to mutants of ANF, preferably mutants of ANF and ANF derivatives, homologs, and fragments, preferably ANF mutants having one or more substitutions of amino acid residues and that binds to an anti-ANF antibody, more preferably an ANF mutant with a mutation at amino acid position 1 (of the rat or human ANF sequences as depicted in FIGS. 2 and 5, respectively (SEQ ID NOS:1 and 4, respectively)), most preferably where the mutation at position 1 of ANF is a substitution of the glycine residue with a serine.
The present invention further provides methods of treating or preventing stroke or ischemic disease, preferably ischemic stroke. In one embodiment, stroke or ischemic disease is treated or prevented by administration of a mutant ANF or a derivative, fragment or homolog thereof, preferably ANF mutants having one or more substitutions of amino acid residues and that bind to an anti-ANF antibody, more preferably an ANF mutant with a mutation at amino acid position 1 (of the rat ANF sequence as depicted in FIG. 2; SEQ ID NO:1), most preferably where the mutation at position 1 of ANF is a substitution of the glycine residue with a serine. Pharmaceutical compositions are also provided.
Also provided are methods of diagnosis, prognosis, and screening by detecting mutant ANF proteins and/or nucleic acids. In a preferred embodiment, an allele protective against stroke is screened for by detecting the presence of a mutation in the ANF gene or protein. Specific oligonucleotides that can be used to detect these mutant alleles are provided. Diagnostic, prognostic and screening kits are also provided.
In another aspect of the invention, assays for screening for ANF mutants that are protective for stroke are provided. Additionally, the invention also includes methods of screening for modulators of ANF activity that affect stroke latency or stroke severity.